Combination of Tyrosine Kinase Inhibitor with β-Catenin/CBP Modulator C82 Reverses TKI Resistance, Eradicates Quiescent CML Stem/Progenitors Cells, and Overcomes MSC-Associated Microenvironmental Protection
Authors: Zhou Hongsheng , Mak Po Yee , Mu Hong , Mak Duncan H , Kouji Hiroyuki , Konopleva Marina , Cortes Jorge E. , Andreeff Michael , Carter Bing Z 
Published: October 2019

Abstract
Bcr-Abl tyrosine kinase inhibitors (TKIs) are effective in inducing remissions and improving survival in patients with CML but do not eliminate CML leukemia stem cells (LSCs). Wnt/β-catenin pathway is established to be active in CML and essential for CML LSC, while adult HSCs do not require fully active β-catenin for maintenance. Furthermore, Wnt/β-catenin signaling pathway plays a critical role in TKI resistance and stromal-mediated microenvironmental protection for CML stem and progenitor cells. We propose that combinations of β-catenin inhibitors and TKIs represent a potentially effective therapy by targeting both CML LSCs and leukemia-mediated microenvironmental protection. C82 is a novel β-catenin/CBP modulator that via binding to CBP inhibits the interaction of β-catenin and CBP and thus disrupts Wnt/β-catenin/CBP mediated cell proliferation and self-renewal signaling.
CML cell lines and primary CML-BC patient samples were treated with combinations of C82 with different TKIs, including imatinib (IM), nilotinib (NIL), dasatinib (DAS), and ponatinib (PON). Both TKI-sensitive KBM5 (IC50=0.50±0.06µM, EC50=0.32±0.01µM, 48h) and TKI-resistant KBM5-STIT315I (IC50=1.44±0.06µM, EC50=0.36±0.09µM, 48h) cells were sensitive to C82. C82-TKI combinations synergistically induced apoptosis (C82-NIL, CI=0.30±0.07 and C82-DAS, CI=0.20±0.01 in KBM5; C82-NIL, CI=0.24±0.09 and C82-DAS, CI=0.36±0.05 in KBM5-STIT315I at 48h; respectively) and inhibited cell grwoth in both cell lines. KBM5, KBM5-STIT315I and K562 were co-cultured with normal human bone marrow derived-MSCs. Western blot showed that CML/hMSCs co-cultures increased β-catenin, CD44, and survivin proteins in CML cell lines. C82-TKI combinations induced similar degrees of cell death and proliferation inhibition with or without hMSC co-cultures, indicating the combination strategy can overcome MSC-mediated microenvironmental chemoprotection in CML. Western blot analysis showed that C82 significantly inhibited CD44 and survivin expression which was further reduced by C82-TKI combinations in KBM5 and KBM5-STIT315I cells.
C82-TKI combinations were evaluated in CML sample (n=6) from heavily-treated and TKI-resistant CML-BC patients. Four out of 6 sample harbored BCR-ABL kinase mutations, including T315I, E255K/V, and H396R. Mononuclear cells from the patients were stained with cell division tracking dye CFSE and then co-cultured with hMSCs. Flow cytometry was performed to identify CD34+CFSEbright and CD34+CFSEdim cells, as quiescent and proliferating population, respectively. When CML cells were treated without hMSC co-culture, C82-TKI combinations exerted stronger synergistic effects in CFSEbright quiescent cells (CI=0.21±0.06, 0.29±0.07, 0.48±0.15, or 0.26±0.03 for combination of C-82 with IM, NIL, DAS, or PON) compared with CFSEdim proliferating cells (CI=0.43±0.05, 0.43±0.17, 0.50±0.20, or 0.44±0.06 for combination of C-82 with IM, NIL, DAS, or PON; respectively). While under co-culture conditions, similar levels of synergy was observed in proliferating (CI=0.39±0.02, 0.23±0.02, 0.32±0.05, or 0.27±0.01 for combination of C-82 with IM, NIL, DAS, or PON) and quiescent cells (CI=0.23±0.02, 0.20±0.01, 0.39±0.10, or 0.20±0.04 for combination of C-82 with IM, NIL, DAS, or PON; respectively). C82-TKI combinations also synergistically induced cell death in CD34+38- CML cells (n=4) and yielded minimum effect on normal bone marrows CD34+ cells (n=3). Invivo studies are ongoing with immunodeficient NOD/SCID/IL2rγnull mice injected with CML-BC patient samples. An open-label, dose-escalation phase I/II study of PRI-724 (active metabolite of C82) for advanced myeloid malignancies (NCT01606579), including CML patients in combination with dasatinb, is enrolling patients at MD Anderson Cancer Center and other centers.
Our data demonstrate that β-catenin/CBP signaling pathway plays a critical role in quiescent CML stem/progenitor cells and disruption of the β-catenin/CBP interaction with C82 could overcome MSC-mediated microenvironmental protection for not only proliferating but also quiescent stem/progenitor cells in CML. Combinations of β-catenin/CBP signaling pathway modulator C82 with TKIs represent a potentially promising strategy to tackle TKI resistance and eradicate CML stem/progenitors cells and should be further investigated in larger studies.

Disclosures
Kouji: PRISM Pharma Co., Ltd: Employment. Cortes:PRISM Pharma Co., Ltd: Clinicaltrial PI for NCT01606579 Other. Andreeff:PRISM Pharma Co., Ltd: Research Funding. Carter:PRISM Pharma Co., Ltd: Research Funding.