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Letters to the Editor
CD34+-selected cells from five AML patients were treat- ed with WNT974 or the control (DMSO) and used in colony-forming unit (CFU) assays. We found no signifi- cant differences in the number of colonies in primary CFU after 2 weeks of culture (Figure 2A, Online Supplementary Figure S1B, left panels). Primary colonies were harvested and then re-plated in methylcellulose for an additional 14 days. We found significant decreases in the number of secondary colonies in WNT974-treated cells compared with cells treated with the control, sug- gesting that WNT974 targeting has an impact on LSC self-renewal (Figure 2A, Online Supplementary Figure S1A, right panels). We performed the same experiment using bone marrow from three adult healthy donors and CD34+ cells from three cord blood specimens. As shown in Figure 2B and Online Supplementary Figure S1C, the treatment did not affect normal cells.
Next, we analyzed whether WNT974 has a role in reg- ulating LSC quiescence by using cell membrane labeling retention assays. For these experiments, CD34+-selected samples from three AML patients were stained with cell trace violet (CTV) dye, which incorporates into the cell membrane and can be detected by flow cytometry. CD34+-selected AML patients’ cells were isolated, labeled with CTV, and treated for 3 days with WNT974 and control. The number of viable (7-AADneg), quiescent (CTVhigh/CD34+) cells was then determined by flow cytometry. CTVhigh/CD34+ cells have been shown previ- ously to increase leukemia-initiating ability, and enhanced engraftment in NSG mice. However, in all ana- lyzed patients’ samples, we did not find a significant decrease in the number of CTVhigh/CD34+ cells after treat- ment with WNT974, compared with controls, except for patient 2 at the highest WNT974 dose (t-test; P=0.0077) (Figure 2C). In order to assess whether the treatment can affect the bulk cell population, we performed an annex- in-V/propidium iodide assay on the same CD34+ AML patients’ samples treated with WNT974 after 72 h, and found only a slightly increase of apoptosis at the higher concentration (Figure 2D).
Finally, we investigated whether WNT974 could also effectively target LSC in vivo. To test the effects of WNT974 in vivo, we used our well-characterized MllPTD/WT/Flt3ITD/WT double knock-in CN-AML mouse model.15 This model develops an aggressive AML with 100% penetrance and leads to death within 5 to 8 weeks in secondary bone marrow transplantation.15 First, we confirmed that WNT974 treatment has a similar effect on the primary murine AML cells in this model by conduct- ing a CFU in vitro assay on CD117+ murine cells from bone marrow of AML mice. We observed that the treat- ment was able to reduce proliferation after the re-plating (Figure 3A) similar to the treatment in human AML cells. Next, we transplanted MllPTD/WT/Flt3ITD/WT leukemic cells (CD45.2+) into lethally irradiated wild-type (WT)-BoyJ (CD45.1+) mice together with whole bone marrow cells
+
from WT-BoyJ donors (CD45.1 ). Two weeks after
engraftment, mice were treated with WNT974 or vehicle (control) at a dose of 5 mg/kg twice a day by oral gavage for 1 week. The design of the experiment is shown in Figure 3B. After the last dose, mice were sacrificed and bone marrow and spleen cells were obtained. We meas- ured the Wnt target gene MYC as a surrogate for Wnt pathway targeting in leukemic cells in vivo. We found sig- nificant downregulation of murine Myc in spleen cells that were used during the primary transplants (Online Supplementary Figure S2A). Next, we determined whether Wnt pathway knock-down by WNT974 affected leukemia engraftment in secondary bone marrow trans-
plantation, a key feature of stem cells. Leukemic donor cells obtained from mice spleen treated with WNT974 or vehicle were transplanted into lethally irradiated BoyJ recipients. Despite observing a reduction of Wnt path- way target gene expression in the spleens of secondary transplanted mice (Online Supplementary Figures S2B-E), we did not observe any differences between overall sur- vival (Figure 3C), or engraftment at 1 month (Figure 3D), after re-transplantation. In conclusion, the data show that WNT974 treatment was able to reduce Wnt path- way activity in leukemic cells and decreased self-renewal of primary AML LSC in vitro. However, WNT974 treat- ment alone was unable to impact LSC functions in vivo. For these experiments, we used high doses of WNT974, higher than the equivalent doses used in human clinical trials with this drug. Thus, it is unlikely that the lack of in vivo effect is due to poor pharmacokinetics or pharma- codynamics. This is supported by the fact that we observed Wnt target downregulation in leukemic cells in the treated mice. Another possibility is that other path- ways or even Wnt reactivation could compensate for the initial Wnt downregulation in LSC, and this could be driven by the microenvironment. It is likely that a com- bination of WNT974 with other active agents in AML could lead to sustained targeting and eradication of LSC in AML as was shown in chronic myeloid leukemia.14
Felice Pepe,1 Marius Bill,1 Dimitrios Papaioannou,1 Malith Karunasiri,1 Allison Walker,1 Eric Naumann,1 Katiri Snyder,1 Parvathi Ranganathan,1,2
Adrienne Dorrance1,2 and Ramiro Garzon1,2
1The Ohio State University Comprehensive Cancer Center and 2Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
Correspondence:
RAMIRO GARZON - ramiro.garzon@osumc.edu. doi:10.3324/haematol.2020.266155
Received: July 7, 2020.
Accepted: September 8, 2021. Pre-published: September 16, 2021.
Disclosures: no conflicts of interest to disclose.
Contributions: RG conceived the idea; RG and FP designed the study; FP, MK and EN performed the in vitro experiments; FP, MB and KS performed the in vivo experiments; FP, DP, PR, AD and RG analyzed and interpreted the data; RG supervised the study; FP, AD and RG drafted the manuscript.
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