Inhibition of CML Stem Cell Renewal By the Porcupine Inhibitor WNT974
Authors: Agarwal Puneet , Zhang Bin , Ho Yinwei , Cook Amy , Li Ling , Wang Youzhen , Mclaughlin Margaret E , Bhatia Ravi 
Published: October 2019

Abstract
Treatment of chronic myeloid leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKIs) fails to eradicate the leukemia stem cells (LSCs) from which the disease arises. Others and we have shown that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our studies indicate that microenvironmental Wnt signaling may play a role for in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Wnt secretion and activity requires their palmitoylation by the Porcupine acyltransferase (PORCN). WNT974 is a potent PORCN inhibitor that inhibits Wnt signaling and demonstrates in vivo efficacy against Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). We have investigated whether WNT974 could sensitize CML stem/progenitor cells to TKI treatment. We observed that CML CD34+ cells show enhanced increased phospho-LRP6 (ser1490) expression, β-catenin protein levels, and colony-forming cell (CFC) growth following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells (isolated from cord blood of healthy donors) indicating an enhanced Wnt signaling response. We observed significant upregulation of FZD4 and FZD5 expression in CML compared to normal CD34+ cells on qPCR analysis, which could potentially contribute to their enhanced Wnt sensitivity. CML CD34+ cells enhanced Wnt signaling following co-culture with human bone marrow mesenchymal stromal cells (MSC) as evidenced by increased β-catenin expression and nuclear translocation, and increased expression of the Wnt target genes c-Myc, Cyclin-D1, PPARδ, and Axin2. WNT974 exposure reduced Wnt secretion from MSC and inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with WNT974 (1μM) in combination with nilotinib (Nil, 1μM) resulted in significant inhibition of CML CFC growth, both in the presence and absence of MSC, and to a significantly greater extent than the normal CFC growth. Although, no difference in engraftment of normal CD34+ cells treated with individual drugs or combination in immunodeficient mice after 16 weeks was observed, treatment of CML CD34+ cells with WNT974 + Nil resulted in significantly decreased engraftment of BCR-ABL+ CML LSC. We further investigated the effects of WNT974 and Nil on CML hematopoiesis in vivo using a transgenic BCR-ABL mouse model of CML. BM cells from CML mice were transplanted into congenic wild-type FVB/N mice to generate CML-like disease in recipient mice. Three weeks after transplantation, mice were treated with Vehicle, WNT974 (5mg/kg bid p.o), Nil (50mg/kg qd p.o), or the combination for 2 weeks. Treatment with WNT974 + Nil resulted in significantly greater reduction in WBC levels, %neutrophils and myeloid cells (Gr-1+ CD11b+) in the peripheral blood of CML mice compared with Nil alone. Long-term hematopoietic stem cells (LTHSC) and committed progenitors (MPP, CMP, GMP) were significantly reduced in the BM and spleen of mice treated with combination compared to individual drugs. Next, we evaluated subsequent survival of mice after completion of 3 weeks of drug treatment. Whereas all control mice died by 30 days, mice treated with the combination of WNT974 + Nil showed significantly prolonged survival after discontinuation of treatment when compared to individual drug-treated mice (p=0.0146). To assess the effects on LSC self-renewal potential, BM cells from primary drug-treated mice were transplanted into normal secondary recipient mice. At 12 weeks post-transplantation, there was a significant reduction in LTHSC, MPP, and GMP populations in the BM of mice transplanted with cells from primary mice treated with the combination compared with individual drugs, indicating reduced LSC self-renewal. Finally, we evaluated the effect of in vivo treatment on Wnt/β-catenin signaling in leukemic cells. Significant downregulation of c-Myc, Cyclin D1, and Axin2 was seen on qPCR performed on c-Kit+ BM progenitors from mice treated with WNT974 and Nil+WNT974 compared to vehicle treated mice. Therefore, we conclude that inhibition of Wnt secretion and signaling by treatment with the PORCN inhibitor WNT974 effectively and selectively inhibits the self-renewal capacity of CML LSC. These results support the further evaluation of combinations of PORCN inhibitors with TKI treatment for selective targeting of CML LSC.

Disclosures
Wang: Novartis Institute of Biomedical Research: Employment. Mclaughlin:Novartis Institute of Biomedical Research: Employment.