The PI3K/mTOR inhibitor PF-04691502 induces apoptosis and inhibits microenvironmental signaling in CLL and the Eµ-TCL1 mouse model by Matthew D. Blunt, Matthew J. Carter, Marta Larrayoz, Lindsay D. Smith, Maria Aguilar-Hernandez, Kerry L. Cox, Thomas Tipton, Mark Reynolds, Sarah Murphy, Elizabeth Lemm, Samantha Dias, Andrew Duncombe, Jonathan C. Strefford, Peter W. M. Johnson, Francesco Forconi, Freda K. Stevenson, Graham Packham, Mark S. Cragg, and Andrew J. Steele Blood Volume 125(26):4032-4041 June 25, 2015 ©2015 by American Society of Hematology

Inhibition of mTOR augments AKT signaling in CLL, which can be overcome by a dual PI3K/mTOR inhibitor. Inhibition of mTOR augments AKT signaling in CLL, which can be overcome by a dual PI3K/mTOR inhibitor. (A) CLL cells were pretreated with everolimus (1 μM) or PF-04691502 (1 μM) for 2.5 hours prior to treatment with soluble anti-IgM (IgM) for 15 minutes. Immunoblotting was performed for phosphorylated AKT (pAKTS473), S6 kinase (pS6KT389), and S6 (pS6 ribsosomal subunitS235/236). Bcl-2 was used as a loading control. The fold change in (B) pAKTS473 (n = 7), (C) pS6KT389 (n = 6), (D) pS6S235/236 (n = 7), and (E) AKTT308 (n = 5) compared to the untreated basal control after the various treatments described in panel A. *P < .05, **P < .01. Error bars represent SEM. NA, untreated basal control; SEM, standard error of the mean. Matthew D. Blunt et al. Blood 2015;125:4032-4041 ©2015 by American Society of Hematology

PF-04691502 reduces cell viability of CLL cells independently of prognostic markers. PF-04691502 reduces cell viability of CLL cells independently of prognostic markers. (A) CLL samples (n = 25) and normal B (CD19+) and T cells (CD3+) (n = 5) were treated in the presence or absence of PF-04691502 (0.0049-40 μM) for 24 hours. Cell viability was calculated using annexin V/PI assays and measured by flow cytometry. For comparison, a proportion of the same CLL samples were treated with idelalisib (0.0049-40 μM) (n = 12). (B) CLL samples (n = 6, 3 low IC50 and 3 high IC50 at 24 hours) were treated with PF-04691502 (0.0025-2.5 μM) for 24 to 72 hours. Correlation of the response to PF-04691502 with prognostic markers (C) IGHV (n = 21), (D) ZAP-70 (n = 23), and (E) CD38 (n = 23). Error bars represent SEM. M-CLL, mutated IGHV genes; U-CLL, unmutated IGHV genes; +ve, positive; -ve, negative. Matthew D. Blunt et al. Blood 2015;125:4032-4041 ©2015 by American Society of Hematology

PF-04691502 induces the intrinsic apoptosis pathway after PI3K/mTOR pathway inhibition. PF-04691502 induces the intrinsic apoptosis pathway after PI3K/mTOR pathway inhibition. (A) Immunoblotting was used to show expression of the Bcl-2 family of proapoptotic proteins, namely Noxa and Puma, in resting nonactivated cells. pAKTS473 and pS6KT389 were used to confirm that PF-04691502 treatment (18 hours) had inhibited the PI3K/mTOR pathway. HSC70 was used as a loading control. The figure is representative of 9 independent experiments. (B) BAX conformational change was investigated in the presence or absence of PF-04691502; immunoprecipitation (IP) with the BAX 6A7 antibody was performed as described in supplemental Methods). IgH and IgL were included to confirm that identical quantities of antibody were used for the immunoprecipitation. Blot is representative of 4 independent experiments. (C) Cleavage of caspase 3 and its substrate PARP (marker of apoptosis). Blot is representative of 10 independent experiments. (D) CLL samples (n = 7) were treated with PF-04691502 as above but with or without the pan-caspase inhibitor ZVAD.fmk and analyzed by flow cytometry for annexin V/PI negativity. (E) Stromal fibroblasts (HFFF2) were cocultured with CLL cells and the whole well was treated in the presence or absence of continuous PF-04691502 (2.5 μM) for 24 hours. CLL cells were removed by scraping (or by pipetting; see supplemental Figure 3D), and cell viability was analyzed using annexin V/PI analysis by flow cytometry (n = 7). (F) The assay was performed as in panel E, except that the CLL cells were pretreated for 1 hour with PF-04691502 prior to washing out the drug, and the treated CLL cells were plated into wells containing stromal fibroblasts (n = 7) (described in supplemental Methods). Error bars represent SEM. No difference was observed between scraping all cells (panel E) or washing off only the CLL cells (see supplemental Figure 3D). *P < .05, **P < .01. NS, not significant. Matthew D. Blunt et al. Blood 2015;125:4032-4041 ©2015 by American Society of Hematology

PF-04691502 inhibits anti-IgM–induced signaling. PF-04691502 inhibits anti-IgM–induced signaling. CLL cells were treated with (A) soluble anti-IgM beads (n = 9) and (B) immobilized (Imm) anti-IgM beads (n = 5) prior to evaluation of downstream signaling in the presence and absence of PF-04691502 (0.0025-2.5 μM) by immunoblot analysis. Representative cases are shown. CLL cells were treated for 1 hour with PF-04691502 prior to soluble/immobilized anti-IgM addition. pAKTT308 and pS6KT389 were used as markers of PI3K signaling, pAKTS473 and pS6S235/236 were used as markers of mTOR signaling, and pERKT202/Y204 was used as a marker of mitogen-activated protein kinase signaling. HSC70 was used as a loading control. Matthew D. Blunt et al. Blood 2015;125:4032-4041 ©2015 by American Society of Hematology

PF-04691502 inhibits CXCL12 signaling and subsequent migration. PF-04691502 inhibits CXCL12 signaling and subsequent migration. (A) CLL cells were treated with 200 ng/mL CXCL12 for the times indicated in the presence or absence of PF-04691502 (0.25-2.5 μM). PF-04691502 was added 30 minutes prior to the addition of CXCL12. Immunoblotting was performed for pAKTS473, pS6KT389, pS6S235/236, pERKT202/Y204, total proteins, and the loading control HSC70. Blot is representative of 6 independent experiments. (B) Using a transwell migration assay, we evaluated the migration of CLL cells toward 200 ng/mL CXCL12 in the presence or absence of PF-04691502 (1.25-2.5 μM). Flow cytometry was used to count the number of CD5+CD19+cells that had passed through the transwell filter (n = 5) (see supplemental Methods). Error bars represent SEM. *P < .03. Matthew D. Blunt et al. Blood 2015;125:4032-4041 ©2015 by American Society of Hematology

PF-04691502 inhibits anti-IgM– and CXCL12–induced signaling in Eμ-TCL1 murine cells. PF-04691502 inhibits anti-IgM– and CXCL12–induced signaling in Eμ-TCL1 murine cells. Eμ-TCL1 cells were removed from mice and grown in vitro. Eμ-TCL1 cells were treated with soluble (Sol) anti-IgM (5-15 minutes) (A) or 200 ng/mL CXCL12 (B) in the presence or absence of PF-04691502 (0.0025-2.5 μM), and the effects on downstream signaling (pAKTT308, pAKTS473, pS6KT389, pS6S235/236, and pERKT202/Y204) were investigated by immunoblotting. HSC70 was used as a loading control. (C) Viability of Eμ-TCL1 cells by PF-04691502 was evaluated by annexin V/PI assays and represented as percentage viable cells. (D) Migration was performed as previously described in Figure 5B. Error bars represent SEM. *P < .02. Matthew D. Blunt et al. Blood 2015;125:4032-4041 ©2015 by American Society of Hematology

The effect of PF-04691502 in the Eμ-TCL1 mouse model. The effect of PF-04691502 in the Eμ-TCL1 mouse model. Mice inoculated with Eμ-TCL1 tumor cells were treated by oral gavage with 5 or 10 mg/kg/d PF-04691502 or placebo control over 14 days of continual treatment after the emergence of a leukemic phase occurred at day 21 (depicted by the arrow in panel A). (A) Peripheral leukemic (CD5+CD220+) cell number was evaluated by flow cytometry in the presence of PF-04691502 or the vehicle control. (B) Illustration of the spleen sizes from mice treated with 10 mg/kg/d PF-04691502 or vehicle control as compared to the wild-type mouse C57BL/6. (C) Weights of the spleens depicted in panel B. (D) Leukemic cell number was also investigated in various organs, including spleen (SPL), lymph nodes (LN), and bone marrow (BM) after treatment with PF-04691502 or the vehicle control. Error bars represent SEM. *P < .02, **P < .004, ****P < .0001. PF, PF-04691502. Matthew D. Blunt et al. Blood 2015;125:4032-4041 ©2015 by American Society of Hematology