Disruption of NOTCH1-MYC-CD44 Axis Targets Leukemia Initiating Cells (LIC) in T-ALL

Abstract

Background:Persistence of leukemia initiating cells (LIC) in T acute lymphoblastic leukemia (T-ALL) results in relapse in 20- 25% of pediatric and over 50% of adult patients1. LIC are characterized by high CD44 expression and low reactive oxygen species (ROS) levels 2, 3. T-ALL LIC maintain low ROS by cystine/glutamate anti-porter complex of which CD44 is a key component 2, 4. CD44 also interacts with microenvironmental components; hyaluronic acid, osteopontin, fibronectin etc. NOTCH1 transcriptionally upregulates CD44/MYC by binding the upstream ‘super-enhancer’ sites. BRD4, a member of the bromodomain and extra terminal domain (BET) family, is a critical scaffold of super-enhancer complexes that binds acetylated histones (3 and 4) and drives NOTCH1 mediated MYC/CD44 transcription 5, 4, 1. We hypothesized that degradation of BRD4 with hetero-bifunctional PROteolysis TArgeting Chimera (PROTACTM) such as ARV-825, will lead to efficient and sustained downregulation of NOTCH1/MYC /CD44 transcription and disrupt cell intrinsic and extrinsic pathways for persistence of T-ALL LIC.Methods: We confirmed the anti-leukemia effect of ARV-825 (IC50 pico to low nanomolar) against T-ALL cells including early T-cell phenotype (ETP) and Gamma-secretase inhibitor (GSI) resistant T-ALL8. To confirm disruption of NOTCH1/MYC/CD44 axis in-vivo, we specifically tested the impact of BRD4 degradation on NOTCH1 and its target genes including MYC, CD44 and as functional readout, intra-cellular ROS, in a T-ALL/ patient-derived xenograft (PDX) mouse model of disseminated leukemia possessing a constitutively active NOTCH1 mutation. Mass cytometry based proteomic analysis (CyTOF) studies were done to quantitatively assess T-ALL LICs and suppression of NOTCH1-MYC-CD44 axis, and secondary transplantation was carried out into sub-lethally irradiated mouse recipients to functionally evaluate LIC elimination.Results: ARV-825 mediated sustained BRD4 degradation resulted in profound down-regulation of MYC, CD98, CD44 and its variants (CD44v). Moreover, we observed down-regulation of cell intrinsic anti-apoptotic proteins Bcl-2, Bcl-XL. As a functional correlate of down-regulation of CD98/CD44/CD44v (glutathione anti-porter system), flow cytometry confirmed increased intracellular ROS and decreased reduced glutathione (GSH).In a PDX mouse model of human T-ALL, ARV-825 treatment improved survival compared to mice treated with vehicle (P=0.002) (Fig.1). CyTOF analysis of mouse bone marrow cells showed quantitative reduction of phenotypically defined LIC (CD4+CD8+CD7+ CD19- ) 6,7 with down regulation of the NOTCH1-MYC-CD44 axis along with oncogenic molecules (transcription factors Myc and NFkB, cell cycle regulators, activated PI3K/Akt, and anti-apoptotic Bcl2 family proteins) in mice treated with ARV-825 (Fig.2). Finally, secondary transplantation of equal number of human CD45+ cells from Vehicle and ARV-825 treated mice in to NSG mice led to delayed leukemia development and extended survival of mice engrafted from ARV-825 treated mice (Vehicle:38 days Vs ARV-825: 58 days P=0.0001/ Vehicle:36.5 days Vs ARV-825: 50 days P=0.0001) (Fig.3), providing functional confirmation of LIC elimination.Conclusion: Degradation of BRD4 with PROTAC (ARV-825), modulates the NOTCH1/MYC/CD44 axis and has the potential of therapeutically targeting the LIC in T-ALL.ReferencePui CH, Carroll WL, Meshinchi S, Arceci RJ. Journal of clinical oncology 2011; 29(5): 551-565.Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H et al.Cancer cell 2011; 19(3): 387-400.Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN et al.Nature 2009; 458(7239): 780-783.Garcia-Peydro M, Fuentes P, Mosquera M, Garcia-Leon MJ, Alcain J, Rodriguez A et al.The Journal of clinical investigation 2018. doi: 10.1172/JCI92981Sanchez-Martin M, Ferrando A. Blood 2017; 129(9): 1124-1133.Cox CV, Martin HM, Kearns PR, Virgo P, Evely RS, Blair A. Blood 2007; 109(2): 674-682.Gerby B, Clappier E, Armstrong F, Deswarte C, Calvo J, Poglio S et al.Leukemia 2011; 25(8): 1249-1258.Sujan Piya, Hong Mu, Seemana Bhattacharya, Teresa McQueen, R. Eric Davis, Vivian Ruvolo, Natalia Baran, Yimin Qian, Craig M. Crews, M. James You , Patrick Zweider-McKay, Marina Konopleva, Hagop Kantarjian , Michael Andreeff1, Gautam Borthakur. 59 th Annual Meeting &Exposition, Atlanta GA: December 9-12, 2017. DisclosuresQian:Arvinas LLC Inc: Employment. Raina:Arvinas LLC Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Research Funding.