Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells (original) (raw)
Decreased leukemic colony formation. We bred Pik3r1f/fPik3r2–/– (12, 13) with CD19-Cre mice to create mice that have Pik3r1 (p85α/p55α/p50α) deleted specifically in the B cell lineage and Pik3r2 (p85β) deleted in all cells. We harvested BM from Pik3r1f/fPik3r2+/+, Pik3r1f/fPik3r2–/–, Pik3r1_Δ/Δ_Pik3r2+/+, and Pik3r1_Δ/Δ_Pik3r2–/– mice and assessed transformation efficiency following infection with a retrovirus expressing the p190 isoform of BCR-ABL (p190), together with either GFP or human CD4 lacking the cytoplasmic tail (hCD4). For simplicity, we refer to the B lineage progenitors derived from these mice as WT, β-null, α-null, and α/β-null cells, respectively.
β-null progenitors were equivalent to WT cells in the number of CFUs (CFU–pre-B; Figure 1A). In both α-null and α/β-null progenitors, we observed a significant decrease in transformation efficiency (~50%) relative to that in controls (Figure 1A). We considered the possibility that deletion of Pik3r1 was incomplete and that cells that delete the floxed Pik3r1 allele have a competitive disadvantage. To test this, we selected single colonies (CFU–pre-B), monitored the expansion of these leukemic colony-forming cells (L-CFCs) in liquid culture, and assessed the deletion by immunoblotting for p85 expression (Figure 1, B and C, and Table 1). Only 33% ± 3% (n = 7 experiments; 69 of 215 total colonies selected) of the α/β-null L-CFCs could be expanded versus 86% ± 5% of WT (n = 3; 44 of 52), 87% ± 4% (n = 7; 113 of 134) of β-null, and 58% ± 6% (n = 4; 51 of 90) of α-null L-CFCs (Table 1). Of the expanded L-CFCs from at least 3 experiments, only 29% ± 5% of the α/β-null cells demonstrated deletion by immunoblot analysis compared with 60% ± 7% of the α-null cells (Table 1). The regulatory isoform encoded by the Pik3r3 gene, p55γ, was not detected in WT or β-null cells but was upregulated in some α-null clones and most α/β-null clones (Figure 1C and Table 1). The predominant outgrowth of clones lacking Pik3r1 deletion and/or p55γ upregulation indicates a strong selective pressure to maintain class IA PI3K for colony survival, particularly when Pik3r2 is absent.
Decreased BCR-ABL–mediated (p190) colony transformation of both α-null and α/β-null progenitor B cells. (A) Plating of p190-infected whole BM (5 × 104 cells) from 3- to 6-week-old mice of the indicated genotypes, on M3630 (CFU–pre-B) medium. *P < 0.05 versus WT; #P < 0.01 versus β-null; †P < 0.001 versus β-null; 1-way ANOVA, n = 3–7 independent experiments; mean values ± SEM are shown. (B) Schematic flowchart depicting the clonogenic expansion and assessment of PI3K expression status in L-CFCs. CFU–pre-B colonies were scored at day 7 after infection, and single colonies were selected and transferred to liquid culture. Outgrowth of p190+ L-CFCs was then quantified (“expansion” was defined as reaching ≥1 × 106 cells), followed by immunoblot assessment of Pik3r1 deletion and p55γ upregulation. Table 1 displays percentages of colonies meeting requirements for expansion, deletion of Pik3r1, and absence of p55γ upregulation. (C) monitoring class IA PI3K subunit expression in expanded L-CFCs. Representative immunoblot shows multiple clones of each genotype with variable loss of p85α and p55γ upregulation. p85α-specific antibody was used to distinguish p85α from p85β. White boxes represent clones that failed to delete p85α or upregulated p55γ. Black boxes represent clones that were used for in vitro and/or in vivo assays. (D) Immunoblot for class IA PI3K catalytic subunits in established L-CFCs. Representative blot for 3 independent clones.
Decreased expansion of α/β-null L-CFCs, with high frequency of incomplete Pik3r1 deletion or p55g upregulation
The outgrowth of nondeleting cells from α/β-null cultures was not due to selection of CD19– progenitors that lack Cre expression, since all clones of each genotype expressed CD19 on the surface (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI33337DS1). most clones of each genotype expressed a mixture of markers of pro-B and pre-B cell progenitors (Supplemental Figure 1B), suggesting that the target cell for transformation is not limiting. Moreover, there was a comparable distribution of B cell progenitor subsets among BM cells 14 hours after infection, at which time the marker-expressing cells were similarly distributed, with mainly pro-B (B220+CD43+IgM–) and fewer pre-B (B220+CD43–IgM–) phenotypes in both β-null and α/β-null samples (Supplemental Figure 1C). The inability of some α-null and most α/β-null L-CFCs to expand in culture is most likely due to ongoing deletion resulting in loss of proliferation or survival. Consistent with this model, ablating p85α expression in Pik3r1f/fPik3r2–/– cells by retroviral expression of Cre caused rapid and significant cell cycle arrest and apoptosis (Figure 2). Acute deletion of Pik3r1 in Pik3r2+/+ cells also induced cell cycle arrest and apoptosis; however, the effect on cell cycle was significantly weaker than observed in Pik3r2–/– cells. Together, the results indicate that B lineage transformation by p190 is almost completely dependent on class IA regulatory isoforms of PI3K, with Pik3r1 and Pik3r2 gene products exhibiting partially redundant functions.
Dependence on continued PI3K signaling for maintenance of p190 L-CFCs. (A) L-CFCs were transduced with MSCV-IRES-human CD8 (Empty) or MSCV-Cre-IRES-human CD8 (Cre) and monitored for deletion of Pik3r1 and PI3K/mTOR signaling by immunoblotting 48 hours after infection. The blot is representative of 2 independent experiments using 3 separate clones. (B) L-CFCs were stained for human CD8 48 hours after infection and subsequently fixed and stained with propidium iodide for DNA content analysis of live gated human CD8+ cells by FACS. Graph shows the percentage of cells cycling (S/G2/M); mean ± SEM; n = 3 clones. *P < 0.05, ***P < 0.001; 1-way ANOVA. (C) Human CD8+ L-CFCs were assayed for apoptosis by annexin V/7AAD staining 48 hours after infection. Cell death is expressed as the percentage (±SEM) of annexin V+ cells to both 7AAD+ and 7AAD– cells; n = 3 clones. **P < 0.01, ***P < 0.001, empty versus Cre-infected; 1-way ANOVA.
Reduced class IA PI3K catalytic subunit expression and function. In established L-CFCs that showed complete Pik3r1 deletion without p55γ upregulation, we observed a reduction of p110α, p110β, and p110δ expression in cells lacking class IA regulatory isoforms, with greater loss of catalytic isoform expression in α/β-null compared with α-null or β-null L-CFCs (Figure 1D). Loss of the catalytic subunits is likely due to reduced stability in the absence of regulatory subunit partners (14).
To assess PI3K activity, we visualized PIP3 in fixed cells using a specific antibody. Control L-CFCs exhibited PIP3 immunostaining with a concentration in the plasma membrane and/or intracellular organelles (Figure 3A and Supplemental Figure 2). Pretreatment with wortmannin blocked the staining in most cells, supporting the specificity of detection. Residual PIP3 production was abolished by pretreatment with wortmannin for longer times (Supplemental Figure 2C). The α/β-null L-CFCs displayed significantly reduced PIP3 staining, resembling wortmannin-treated control cells. Thus, the few viable L-CFCs lacking Pik3r1 and Pik3r2 do not sustain PIP3 production by residual class IA catalytic isoforms or the class IB enzyme.
Lack of PI3K/AKT/FOXO functional signaling output in α/β-null L-CFCs. (A) β-null and α/β-null cells were fixed, permeabilized, and immunolabeled with anti-PIP3 antibodies (orange). Nuclei were stained by DAPI (blue). Indicated cells were pretreated with 50 nM wortmannin 30 minutes prior to fixation. Immunoreactive cells were quantitated by counting 100 cells in images acquired with a 40× objective (represented in the graph) and visualized with a 100× objective (represented in the images). Typical examples of n = 4 clones per genotype, 2 independent experiments (see also Supplemental Figure 2). Scale bars: 10.0 μm. **P < 0.001 versus untreated β-null cells; 1-way ANOVA. (B) Multiple clones of L-CFCs of the indicated genotypes were immunoblotted for class IA PI3K isoforms and assessed for phosphorylation of AKT (p-AKT S473) and its substrates FOXO1 and FOXO3a (p-FOXO1 T24/p-FOXO3a T32, right panel; or p-FOXO1 S256, left panel. Note: Anti–p-FOXO1 (S256) can detect p-FOXO4 (S193), but antibodies are not available to confirm mouse FOXO4 expression. Asterisk indicates representative α/β-null L-CFC clone that upregulated p55γ, with a concomitant increase in AKT activity. (C) p190 L-CFCs of the indicated genotypes were treated for 15 minutes with LY294002 (10 μM) and subsequently immunoblotted for p-AKT (S473) and p-FOXO1/4 (S256/S193; note the overexposure with p-FOXO for detection of low signal). n = 3 experiments using 2 different clones. (D) p190 L-CFCs were infected with retroviruses MSCV-IRES-Thy1.1 (Ctrl), MSCV-FOXO3a-IRES-Thy1.1, or MSCV-FOXO3a.A3-IRES-Thy1.1. Apoptosis was assessed in Thy1.1+ cells by annexin V/7AAD staining 48 hours after infection. Data are representative of 3 independent experiments using 2 different clones.
Most α/β-null clones showed no detectable Akt phosphorylation (p-Akt), and clones with residual p-Akt also showed upregulation of p55γ (Figure 3B). Acute Pik3r1 deletion also abrogated AKT phosphorylation (Figure 2A). Cells that compensated for the loss of Pik3r1 and Pik3r2 by upregulating p55γ were eliminated from our further biochemical and leukemogenic investigations.
Lack of PI3K/AKT functional output. Transcription factors of the forkhead box subgroup O (FOXO) family are important AKT substrates in p190 B-ALL cells (9). In agreement with the lack of p-Akt, α/β-null cells maintained a very low level of phosphorylated FOXO1 and FOXO3a (Figure 2A and Figure 3, B and C), which could not be reduced by pharmacological inhibition of PI3K (Figure 3C). This implies that a PI3K-independent kinase accounts for the residual FOXO phosphorylation. We investigated the sensitivity of α/β-null cells to FOXO-mediated death via overexpression of FOXO3a or a mutant (FOXO3a.A3) that lacks the 3 consensus AKT phosphorylation sites (Figure 3D). The control cells that maintained normal levels of PI3K activity were resistant to WT FOXO3a while remaining sensitive to the PI3K-independent form FOXO3a.A3, as described previously (9). In contrast, the α/β-null cells were equally sensitive to death caused by WT and Akt-resistant mutant FOXO3a. This indicates that in the presence of diminished PI3K output, the residual FOXO phosphorylation by an alternative kinase is unable to inactivate overexpressed FOXO proteins.
Proliferation and cell cycle deficits. We labeled cells with PKH26 to assess proliferation of established α/β-null cells. We found that α/β-null cells partitioned the PKH26 dye at a lower rate in comparison to control cells (Figure 4A). Cell cycle analysis supported these results, showing an increase in the percentage of cells in G1 and a dramatic reduction of cells in S phase. α/β-null clones were also smaller, as assessed by forward light scatter (Figure 4A).
Severe loss of leukemogenic potential of α/β-null L-CFCs. (A) Proliferation and cell cycle in α/β-null L-CFCs were assessed in vitro, where β-null (blue) and α/β-null (red) L-CFCs were labeled with 2 μM PKH26 and subsequently analyzed for dilution of PKH26 every 24 hours (left panel, representative of 2 independent experiments using 2 clones) and cellular size analyzed (right panel; *_P <_ 0.05, 2-tailed, paired Student’s _t_ test) by forward scatter; _n_ = 3 clones. (**B**) Survival analysis of sublethally irradiated NOD/SCID mice transplanted with p190 L-CFCs (1 × 106 cells). EGFP+ (or human CD4+) cells (>98% purity) from BM and spleen were collected to assess class IA PI3K deletion status (immunoblot analysis and leukemia-free survival were adjusted as described in Methods and Supplemental Figure 3 and Supplemental Table 1) (C) Representative immunophenotype and cytospins of β-null leukemic blasts in BM, spleen, and lymph nodes.
Reduced in vivo leukemogenic potential. To assess the in vivo consequences of diminished PI3K/AKT signaling, we examined the tumorigenic capacity of multiple L-CFC clones. In this assay, we used only α/β-null L-CFCs lacking detectable p85 proteins or p55γ. WT (1 clone, n = 4 mice), β-null (5 clones, n = 16), α-null (1 clone, n = 4), and α/β-null L-CFCs (5 clones, n = 23) were injected into sublethally irradiated NOD/SCID hosts. Mice with emerging EGFP+ (or hCD4+) leukemic cells developed lymphadenopathy and splenomegaly, with lymphoblasts exhibiting pro/pre–B-ALL morphology and surface phenotype but lacking myeloid lineage markers (Figure 4C and Supplemental Table 1). Mice receiving WT or β-null cells died from pro/pre–B-ALL after an average of 15 and 13 days, respectively (Figure 4B), whereas mice receiving α-null cells succumbed to disease after an average of 35 days (P = 0.052 vs. WT). α/β-null cells had a significantly reduced capacity to initiate leukemia in vivo (Figure 4B). Among all α/β-null transplanted mice, only 1 (of 23) developed a leukemic disease in which cells retained an α/β-null status by immunoblot analysis. This mouse developed leukemic blasts in the BM, lymph nodes, and spleen with some mature B cell markers: CD19+, IgM+, B220+, CD43–, CD25–, CD24+, BP-1+, IgD–, CD11b–. These blasts displayed detectable p-Akt and prominent upregulation of p55γ (Supplemental Figure 3). The striking block in leukemogenesis and the upregulation of p55γ in the 1 instance of emerging leukemia from α/β-null cells strongly suggest that p190 drives B-ALL in a manner completely dependent on class IA PI3K function. However, the residual proliferation in vitro prompted us to examine the drug sensitivity of control and PI3K-deficient L-CFCs.
The mTOR pathway is a vital signaling node for α/β-null cells. To assess the importance of different signaling mechanisms in the absence of PI3K signaling output, we measured cell cycle progression after treatment with pathway-selective inhibitors. Wortmannin, used at a concentration (50 nM) that inhibits all classes of PI3K but not mTOR (15, 16), caused a modest increase in the G1 fraction and a significant decrease in cycling ability (percentage of cells in S/G2/M phase) of control cells (β-null) (Figure 5A, right panel, P < 0.001). However, wortmannin had no significant effect on the cycling ability of α/β-null cells, indicating that other PI3K subclasses do not compensate appreciably for class IA PI3K in cell cycle control. LY294002, a global PI3K inhibitor that also inhibits mTOR directly (15, 17), induced G1 arrest in both control and α/β-null cells; moreover, the α/β-null cells were hypersensitive, as shown by the greater decrease in the number of cycling α/β-null compared with control cells (P < 0.05). Consistent with the interpretation that the effects of LY294002 were mainly due to direct effects on the rapamycin-sensitive mTOR complex–1 (mTORC1), addition of rapamycin also caused a greater decrease in cycling α/β-null cells compared with control cells (P < 0.01). These findings indicate that residual mTORC1 signaling in α/β-null cells is required for proliferation in the absence of class IA PI3K activity. The finding that rapamycin induced greater apoptosis in the α/β-null cells, and that LY294002 caused apoptosis in cells of both genotypes, indicates that only the combined loss of the PI3K/AKT and mTORC1 pathways can lead to a substantial level of cell death in this system. Loss of PI3K/AKT signaling output also rendered p190 cells more sensitive to apoptosis caused by imatinib (Figure 5, A and B).
α/β-null L-CFCs show altered drug sensitivity. (A) L-CFCs were treated with the indicated inhibitors for 24 hours and assessed for apoptosis (subdiploid DNA, left panel) and cycling cells (% S/G2/M phase, right panel) by flow cytometry. Differences in treatment effects between β-null and α/β-null were analyzed by 2-way ANOVA (*P < 0.05, **P < 0.01) and 1-way ANOVA for wortmannin effects compared with untreated controls (§P < 0.001). Mean values ± SEM are shown; n = 4, using 4 different clones. (B) L-CFCs (blue, β-null; red, α/β-null) were treated with or without 1 μM or 2 μM imatinib for 16 hours or 24 hours and analyzed for apoptosis. Cells that stained positive for annexin V and negative or positive for 7-AAD are reported as mean percentage ± SEM; n = 3 independent experiments.
Considering that α/β-null cells were hypersensitive to rapamycin, we examined the status and drug sensitivity of signaling events downstream of mTORC1, including potential feedback regulation of Akt. The serine/threonine kinase mTOR is regulated by both PI3K/AKT and other inputs (18, 19). We found that genetic or pharmacological inhibition of PI3K only partially reduced mTORC1 function as assessed by phosphorylation of ribosomal protein S6 (p-rS6) (Figure 6, A–D; also see Figure 2A for acute Pik3r1 deletion). The residual p-rS6 was comparable to β-null cells treated with the pan-PI3K inhibitor wortmannin. Rapamycin suppressed p-rS6 almost completely in control and α/β-null cells, confirming the presence of PI3K-independent inputs to mTORC1 activity in these cells. Rapamycin also greatly reduced the fraction of phosphorylated 4EBP-1, a direct mTORC1 substrate (Figure 6, B and C), whereas 4EBP-1 phosphorylation was partly maintained in α/β-null cells or wortmannin-treated controls.
α/β-null L-CFCs maintain mTOR signaling via class IA PI3K–independent mechanisms. (A) p190 L-CFCs were treated for 2 hours with the indicated inhibitors, and the percentage of p-rS6 was quantified by ImageJ (upper p-rS6 band was normalized to β-actin; representative of 4 independent experiments and multiple clones). (B and C) L-CFCs were treated with the indicated inhibitors and/or leucine starved for 2 hours and immunoblotted with the indicated antibodies. n = 3, using 3 clones. 3-MA, 3-methyladenine. (D) Cells of the indicated genotypes were starved in leucine-deficient medium (containing 10% dialyzed FCS) for the indicated times or treated with rapamycin (1.5-hour treatment). Control samples were incubated with leucine-sufficient medium (with matched dialyzed serum). n = 3, using 2 clones.
The α/β-null cells lacked p-Akt, while the control cells showed p-Akt that was sensitive to wortmannin, LY294002, and imatinib (Figure 6, A and B). Rapamycin caused an increase in Akt phosphorylation in control cells, with an accompanying increase in AKT activity as judged by FOXO phosphorylation (Figure 6, A–C). This “AKT rebound” phenomenon has been described in many cell contexts and is partly attributed to feedback inhibition of PI3K/AKT signaling by S6 kinases (S6Ks), which is prevented by rapamycin (18–20). A significant consequence of deleting both Pik3r1 and Pik3r2 was the complete loss of rapamycin-induced enhancement of Akt phosphorylation. This might explain the greater sensitivity of α/β-null cells to cell cycle arrest and death following rapamycin treatment (Figure 5A).
Class IA–independent mechanisms for mTOR pathway activation. To explore the mechanism of PI3K/AKT-independent mTOR activity, we assessed the role of 2 potential mTOR regulatory pathways: ERK activity and amino acid sensing. Basal ERK phosphorylation was consistently elevated in α/β-null L-CFCs and blocked by treatment of cells with the MEK inhibitor U0126 (Figure 6C). However, MEK inhibition did not alter mTOR activity as judged by phosphorylation of 4EBP-1 (Figure 6C). p-rS6 expression was modestly reduced in both control and α/β-null L-CFCs, likely due to stimulatory effects of ERK on S6K (21). We assessed the contribution of amino acid sensing by withdrawal of leucine from the culture media. Under these conditions, mTOR activity was rapidly extinguished in α/β-null L-CFCs and to a lesser degree in control cells (Figure 6, C and D), as in other cell systems (16, 22). Amino acid sensing by mTOR is promoted by class III PI3K (hVPS34), an enzyme whose activity is sensitive to wortmannin (22). This might explain the partial inhibition of mTOR signaling by wortmannin in α/β-null L-CFCs that lack class IA PI3K (Figure 6, A–C). Moreover, the class III PI3K inhibitor 3-methyladenine (3-MA) reduced mTOR activity (Figure 6C). In summary, residual mTORC1 activity in α/β-null L-CFCs is ERK independent and sustained by amino acid sensing and other pathways that remain to be defined.
Multitargeted inhibition of pre–B-ALL proliferation with BCR-ABL kinase inhibitor and dual PI3K/mTOR kinase inhibitor. The residual proliferation and mTORC1 signaling in α/β-null cells, together with the feedback activation of Akt in rapamycin-treated control cells, suggested that dual targeting of PI3K and mTOR might be more effective than single targeting of either signaling node. A small molecule inhibitor, PI-103, selectively blocks the kinase activity of class I PI3K catalytic isoforms and both mTOR complexes at nanomolar concentrations (15, 23, 24). We treated p190 L-CFCs (from BALB/c BM) for 48 hours with multiple concentrations of imatinib, rapamycin, or PI-103, alone and in combination (Figure 7A). The percentage of viable cells was determined by MTS assay, and drug synergy was calculated using CalcuSyn software by the method of Chou (25). Both rapamycin and PI-103 suppressed survival with a maximal effect of approximately 50%, though rapamycin was approximately 70-fold more potent on a molar basis (Figure 7A). As previously reported (26), rapamycin showed slight to strong synergy with imatinib, except at lower concentrations of rapamycin (<40% IC50) where the combination produced antagonism (<20% IC50) to near additivity (<40% IC50). PI-103 combined with imatinib exhibited near additivity (<10% IC50) to strong synergy that was consistently greater than observed with rapamycin and imatinib.
Attenuating PI3K and mTOR signaling together with BCR-ABL inactivation provides a greater synergistic response. (A) p190 L-CFCs were treated for 48 hours with the indicated inhibitors alone or in combination with imatinib (IM; left panel). Cell viability and IC50 were determined using the MTS assay. *P < 0.05, imatinib plus PI-103 (PI) versus imatinib plus rapamycin (R); ***P < 0.001, imatinib versus drug combination; repeated-measures 1-way ANOVA. The viability of cells treated with drug combinations at fixed ratios was plotted in the same graph (left panel), and drugs were assessed for synergy by calculating the combination index (CI) using CalcuSyn software (right panel). The CI is plotted as a function of the fractional growth inhibition (CI-Fa plot using median-effect equation; ref. 25) by Monte Carlo simulation of 3 compiled independent experiments. CIs of less than 1, of 1, and of greater than 1 indicate synergism, additive effect, and antagonism, respectively. Mean ± SD is shown; n = 3 independent experiments, using 2 separate clones. (B) p190 L-CFCs were treated for the indicated times with drug combinations, then cell lysates were subjected to immunoblot analysis using 2 gels run and developed concurrently with the same sample set. n = 2. Here we used concentrations of rapamycin (10 nM; ~120% IC50) and PI-103 (250 nM; ~45% IC50) that are achievable in vivo without excessive toxicity (24, 26). Compounds were tested alone or in combination with a clinically achievable concentration of imatinib (2 μM; ~355% IC50).
To define the mechanism of action of drug combinations, we immunoblotted for p-Akt and mTOR activity over a time course of inhibitor treatment (Figure 7B). Rapamycin enhanced p-Akt during the first 6 hours of treatment, though this effect was lost at 24 and 48 hours. Simultaneous inhibition of PI3K and mTOR with PI-103 prevented this feedback rebound, such that p-Akt was effectively suppressed during the first 6 hours. Cells treated with imatinib plus rapamycin showed residual p-Akt that was eliminated in cells treated with imatinib plus PI-103. Also, imatinib plus PI-103 suppressed mTORC1 signaling to a similar extent as imatinib plus rapamycin. Collectively, these results suggest that suppression of rebound Akt activity might explain the greater functional effects of imatinib plus PI-103.
We compared the efficacy of different drug combinations on BCR-ABL–driven leukemic cell expansion in vivo over a 2-day treatment course. BALB/c mice were lethally irradiated and reconstituted with a syngeneic radioprotective graft of BM cells and syngeneic p190 cells (marked by hCD4). After 11 days, when hCD4+ cells could be detected in peripheral blood (Supplemental Figure 4), mice were treated for 2 consecutive days with vehicle alone or suboptimal imatinib (70 mg/kg, twice per day), rapamycin (7 mg/kg, once per day), and PI-103 (40 mg/kg, twice per day), alone or in combination. Mice were then sacrificed, and several aspects of leukemic disease were assessed. Treatment with imatinib alone, compared with vehicle, significantly reduced leukemic disease as measured by spleen weight and percentage of hCD4+ cells in the peripheral blood (Figure 8 and Supplemental Figure 4). Imatinib alone also reduced the number of cycling hCD4+ cells in BM and spleen but caused no significant increase in apoptosis of hCD4+ cells. Rapamycin or PI-103 alone did not significantly reduce spleen weight but did reduce leukemic cell cycling, with variable effects on apoptosis. Imatinib plus PI-103 produced a significantly stronger antileukemic effect than imatinib alone, as measured by spleen weight. In accordance with this, imatinib plus PI-103 caused significantly greater cell cycle arrest and death in leukemia cells compared with imatinib alone. As previously reported (26), imatinib plus rapamycin was also effective and caused a significant increase in apoptosis compared with imatinib alone. However, the reduction in spleen weight and leukemic cell cycling was not statistically greater for imatinib plus rapamycin compared with imatinib alone. Thus, PI-103 but not rapamycin enhanced the effects of imatinib on spleen weight and BM leukemic cell cycling. Note that the PI-103 dose (80 mg/kg/d) was only 11-fold greater than the rapamycin dose (7 mg/kg/d), whereas the IC50 of PI-103 in vitro is nearly 70-fold higher. Overall, these results support the in vitro data in showing that PI-103 is more effective than rapamycin in combining with imatinib to suppress leukemic cell expansion.
Short-term in vivo administration of PI-103 combined with imatinib suppresses leukemic cell expansion more effectively than imatinib alone. (A) Lethally irradiated syngeneic recipients were transplanted with p190 L-CFCs (hCD4+) together with normal BM, then treated on days 11–13 after injection (i.p.; 2 days of treatment) with single and combination doses of imatinib (70 mg/kg, b.i.d.) and rapamycin (7 mg/kg, once per day) or imatinib and PI-103 (40 mg/kg, b.i.d.) or double placebo control (diH2O and 75%/25% DMSO/saline mixture, b.i.d.) prior to sacrifice. Spleen weights after treatment regimen are reported. (B) Percent apoptosis in the BM and spleen was determined by annexin V/7AAD staining of hCD4+ blasts after 2-day treatment regimen. (C) Two hours prior to sacrifice, mice were injected with 5-ethynyl-2′-deoxyridine (EdU; i.p., 1.125 mg) and assayed for cycling cells that incorporated EdU in the BM and spleen with Click-iT EdU AF647 azide and propidium iodide staining (left and right panels). Representative depiction of cycling cells in the BM (left panel). *P < 0.05, ‡P < 0.01, **P < 0.001 versus control; #P < 0.05, †P < 0.01 versus imatinib; 1-way ANOVA; mean values ± SEM are shown; n = 5 mice per treatment group.
To extend these findings from the murine system, we compared the effects of PI-103 and rapamycin on primary human Ph+ leukemias. CD19+CD34+ cells were purified from peripheral blood of patients with Ph+ ALL, PH+ CML with lymphoid BC, or Ph– B-ALL, and clonogenic potential was assessed in the presence of various drug combinations. Ph– B-ALL samples were resistant to imatinib, as expected. In each case of Ph+ disease, the presence of PI-103 combined with imatinib suppressed colony formation to a greater extent than imatinib alone or imatinib in combination with rapamycin; overall, these differences were statistically significant (Figure 9 and Supplemental Figure 5). Notably, the results from samples B and G also indicate that PI-103 can potentiate the antileukemic effect of imatinib in Ph+ samples that are clinically resistant to this ABL inhibitor.
Anticlonogenic effects of PI-103 combined with imatinib in Ph+ leukemia progenitors. Primary Ph+ leukemia samples (Ph+ B-ALLCD34+/CD19+, n = 5; Ph+ CML/ALL-BCCD34+/CD19+, n = 1; and Ph– ALLCD34+/CD19+, n = 3) were assessed for colony formation potential with or without single and combination treatments of imatinib, rapamycin, or PI-103. 100% clonogenic potential was normalized to vehicle-treated samples. Clonogenic frequencies of combination treatments were compared with that of imatinib alone (expressed as percent of imatinib-treated). *P < 0.05, **P < 0.01, ***P < 0.001; 2-way ANOVA; mean values ± SEM are shown; Ph+, n = 5 and Ph–, n = 3, comparing the source of variation from Ph+/– and treatment. Note that in the combination treatments, the imatinib concentration was fixed at its IC50. Cytogeneics, cytospins, and flow cytometric markers of human specimens are given in Supplemental Table 2.