1. AKR1C3 is a biomarker of sensitivity to PR-104 in preclinical models of T-cell acute lymphoblastic leukemia
by Donya Moradi Manesh, Jad El-Hoss, Kathryn Evans, Jennifer Richmond, Cara E. Toscan, Lauryn S. Bracken, Ashlee Hedrick, Rosemary Sutton, Glenn M. Marshall, William R. Wilson, Raushan T. Kurmasheva, Catherine Billups, Peter J. Houghton, Malcolm A. Smith, Hernan Carol, and Richard B. Lock
Blood
Volume 126(10):
September 3, 2015
©2015 by American Society of Hematology

2. In vivo responses to PR-104 of T-ALL and BCP-ALL engrafted mice.
In vivo responses to PR-104 of T-ALL and BCP-ALL engrafted mice. NOD/SCID mice were engrafted with a T-ALL, ALL-27 (A) or a BCP-ALL, ALL-19 (B) patient-derived xenograft. When the proportion of hCD45+ cells in the PB reached 1%, the mice were treated with PR-104 at 200 mg/kg weekly for 2 weeks (red), and compared with vehicle-treated control (black). When the proportion of hCD45 reached 25% in the PB, mice were scored as event on the corresponding Kaplan-Meier curves on the right panel. (C) LGD is plotted for the BCP-ALL and T-ALL xenografts. PR-104 showed a significantly greater efficacy in T-ALL than BCP-ALL xenografts (Fisher exact test, P = .03). (D) COMPARE-like plots show the objective response measures (ORMs) for 4 T-ALL (ALL-8, ALL-27, ALL-29, and ALL-31) (black) and 3 BCP-ALL (ALL-2, ALL-4, and ALL-19) (gray) xenografts. Responses represent the difference in xenograft objective response from the midpoint (0) representative of stable disease (SD). Bars to the right indicate an objective response (PR, CR, and MCR), whereas those to the left indicate PD. Data for the complete panel of xenografts are available in Table 2 and supplemental Figure 1.
Donya Moradi Manesh et al. Blood 2015;126:
©2015 by American Society of Hematology

3. In vitro sensitivity to PR-104A in a panel of BCP-ALL and T-ALL xenografts.
In vitro sensitivity to PR-104A in a panel of BCP-ALL and T-ALL xenografts. Xenograft cells were cultured for 48 hours in increasing concentrations of PR-104A ( μM). Samples were then assayed with resazurin to determine viability based on mitochondrial activity. (A) The IC50 is plotted for the T-ALL and BCP-ALL xenograft panels. (B) Dose response curves of the viability of cells are shown at the different concentrations of PR-104A for the same xenografts that were used in vivo from Figure 1C-D. (C) In vitro and in vivo sensitivity to PR-104A showed a significant correlation in the 7 xenografts examined. Data represent the mean ± SEM of IC50 values for an individual xenograft from a minimum of 3 independent experiments.
Donya Moradi Manesh et al. Blood 2015;126:
©2015 by American Society of Hematology

4. PR-104A sensitivity correlated with AKR1C3 mRNA, protein, and enzymatic activity.
PR-104A sensitivity correlated with AKR1C3 mRNA, protein, and enzymatic activity. (A) A total of 17 ALL xenografts (T-ALL, n = 9; BCP-ALL, n = 8) were profiled on Illumina Human Ref 12 Beadchip arrays, and the top 25 differentially expressed genes between PR-104A–resistant and –sensitive xenografts; genes were ordered based on the P value. Red shows relative upregulation and blue shows relative downregulation. T-ALLs are shown in red and BCP-ALLs in blue. (B) mRNA expression of AKR1C3 by RT-qPCR in the panel of xenografts, and correlated with in vitro IC50 and in vivo LGD. (C) Protein lysates were extracted from T-ALL and BCP-ALL xenografts and AKR1C3 was probed by western blot, quantified, and correlated with IC50 and LGD. A representative immunoblot is shown; for all immunoblots, see supplemental Figure 3. (D) Through a SN34037-sensitive coumberone reduction, AKR1C3 enzymatic activity can be measured by the fluorescent product coumberol. Coumberol formation was measured in vitro in T-ALL and BCP-ALL xenografts, and correlated with in vitro IC50 and in vivo LGD.
Donya Moradi Manesh et al. Blood 2015;126:
©2015 by American Society of Hematology

5. In vivo response to PR-104 in an AKR1C3-overexpressing BCP-ALL xenograft.
In vivo response to PR-104 in an AKR1C3-overexpressing BCP-ALL xenograft. AKR1C3 cDNA was transduced into ALL-11 (BCP-ALL) resulting in higher levels of AKR1C3 protein expression, comparable to levels observed in T-ALL xenografts (A). These cells were then engrafted into NSG mice, and when the proportion of hCD45 cells in the PB reached 1%, mice were treated with PR-104 at 200 mg/kg for 2 weeks. (B) Kaplan-Meier plot shows the event-free survival of mice treated with PR-104 (solid lines) and engrafted with either the AKR1C3 cDNA (blue), the empty vector (orange), or the untransduced xenograft (green). PR-104 treatment significantly increased survival of AKR1C3 overexpressing mice compared with empty vector (Mantel-Cox analysis, P < .0001) Vehicle-treated mice appear in the broken lines. (C) Engraftment was monitored by the proportion of hCD45 cells in the PB of each mouse, and an event was recorded when it reached 25%.
Donya Moradi Manesh et al. Blood 2015;126:
©2015 by American Society of Hematology

6. Bone marrow infiltration of ALL-11 engrafted mice at day 1 and day 8.
Bone marrow infiltration of ALL-11 engrafted mice at day 1 and day 8. Bone marrow infiltration of mice engrafted with ALL-11 empty vector or AKR1C3 cDNA, both of which have GFP reporters. Samples were harvested 24 hours after the first dose of PR-104 (day 1, A) or 24 hours after the second dose of PR-104 (day 8, B) and cells were run on flow cytometry to determine the presence of GFP+ and hCD45+ cell populations. (C) Immunofluorescence for GFP in the bone marrow of PR-104–treated mice at day 8 shows absence of the GFP reporter in mice inoculated with cells transduced with AKR1C3 cDNA compared with empty vector. (D) Quantitative data showing the percentage of hCD45+ cells in n = 8 femurs. Data represent the mean ± SEM. *P < .01; **P ≤ by unpaired Student t test with Welch correction.
Donya Moradi Manesh et al. Blood 2015;126:
©2015 by American Society of Hematology

7. Proportion of human CD45+/GFP+ in the peripheral blood of ALL-11 engrafted mice at event.
Proportion of human CD45+/GFP+ in the peripheral blood of ALL-11 engrafted mice at event. PB from mice engrafted with untransduced (A), empty vector (B), or AKR1C3 cDNA (C) ALL-11 cells was collected when mice reached event (hCD45+ population in blood reaches 25%). One representative mouse is shown from each. The hCD45+ population was gated by flow cytometry to determine the proportion of GFP+ cells circulating in the PR-104–treated (blue) or vehicle (red) groups. The numbers in panels A through C refer to the percentage of GFP− and GFP+ cells in the PR-104–treated mice. (D) Quantification of the proportion of hCD45+/GFP+ cells in all groups.
Donya Moradi Manesh et al. Blood 2015;126:
©2015 by American Society of Hematology

8. AKR1C3 expression correlates with PR-104A sensitivity in primary ALL cells.
AKR1C3 expression correlates with PR-104A sensitivity in primary ALL cells. (A) Mononuclear cells purified from BCP-ALL (solid line) and T-ALL (broken line) patients were treated with PR-104A in coculture with MSC-hTERT cells in a dose response. Cells were stained with 7AAD 24 hours following treatment and percentages of live cells are shown relative to vehicle treatment. (B) Proportion of live cells after treatment with 50 μM PR-104A for 24 hours for BCP-ALL and T-ALL subtypes. (C) RT-qPCR expression of AKR1C3 in patient cells relative to ALL-8 xenograft control. (D) Correlation of AKR1C3 expression and sensitivity to 50 μM PR-104A in BCP-ALL (gray) and T-ALL (black) patient samples. Results shown are the mean of 2 biological repeats. *P < .05; **P ≤ .005 by Mann-Whitney U test.
Donya Moradi Manesh et al. Blood 2015;126:
©2015 by American Society of Hematology