The alarmins, S100A8 (A8) and S100A9 (A9), are low molecular weight proteins belonging to the S100 protein family. Also called calprotectins, A8 and A9 can form homodimers or heterodimers and bind Ca++. A8 and A9 are secreted into the extracellular space and plasma, where they interact with TLR4 (Toll like receptor 4), RAGE (Receptor for Advanced Glycation End products) and CD33. Intracellularly, they act as Ca++ biosensors and activate NADPH oxidase, stimulating ROS production, which activates NLRP3 inflammasome. A8 and A9 also amplify the inflammatory response through TLR4, and induce inflammatory cytokines, including TNFα and IL-6, through activation of NFkB and MAP kinase pathways. A8 and A9 are highly expressed in AML, especially myelomonocytic and monocytic AML, and are associated with poor prognosis. Through scRNA-Seq analysis, A8/A9 heterocomplex was shown to be highly expressed in myeloid and stromal cells and marked progression of bone marrow fibrosis in PMF (Primary Myelofibrosis). In the murine JAK2-V617F-driven PMF model, Tasquinimod (TM, Active Biotech), an orally active, quinoline-3-carboxamide linomide analogue immune-modulatory agent was shown to bind A9, inhibiting its interaction with TLR and RAGE receptors. Notably, TM treatment reduced myeloproliferation, splenomegaly and MF in JAK2-V617F mice. Therefore, based on these findings, in present studies, we determined in vitro and in vivo efficacy of TM against cellular models of advanced MPN cell lines and patient-derived (PD) CD34+ blastic phase (BP, > 5% blasts in PB) MPN cells. Treatment with TM (10 to 30 µM for 96 hours) induced loss of viability in the MPN-AML SET2, HEL92.1.7 cell lines and PD MPN-BP cells, but not in normal CD34+ progenitor cells. RNA-Seq analysis showed that exposure to TM (20 µM for 16 hours) led to negative enrichment of the gene-sets of MYC and E2F targets, of mTORC1 and IL6-JAK-STAT3 signaling, and of inflammatory response in PD MPN-AML cells. This was associated with log2 fold-decline in MYC, BCL2, CDK6, MPL, CCND1 and myeloperoxidase (MPO), but upregulation of mRNA of GFI1 and p21. CyTOF analysis revealed that TM treatment significantly reduced expressions of A8, A9, p-ERK, MPO, CXCR4, Cyclin D1, PU.1 and Ki67, but increased protein levels of GFI1, p21 and cleaved PARP in phenotypically defined PD, CD34+ MPN-BP cells expressing high levels of CLEC12A, CD99, CD123 but low expression of CD11b. Additionally, our findings showed that co-treatment with TM (5 to 30 µM) and ruxolitinib (250 to 1000 nM), BET inhibitor OTX015 (50 to 250 nM) or pelabresib (CPI-0610) (100 to 500 nM), or BCL2/Bcl-xL inhibitor navitoclax induced synergistic lethality in advanced MPN-BP cells exhibiting delta synergy scores of >1.0 (by the ZIP method). Notably, our findings also demonstrated that treatment with TM (10 or 30 mg/kg/day, by oral gavage) for 8-weeks, significantly improved survival without inducing any toxicity in immune-depleted NSG mice engrafted with PDX cells of MPN-AML with mutations in CALR and TERT. In a separate experiment on the same PDX model, treatment with TM (30 mg/kg/day) also induced significantly greater survival advantage than treatment with ruxolitinib (30 mg/kg/day) or OTX015 (30 mg/kg/day) by oral gavage. These findings clearly demonstrate preclinical efficacy of TM in advanced MPN-BP cellular models and create the rationale to further interrogate the efficacy of TM alone and in combinations with current, front-line therapies for advanced MPN with excess blasts.
Disclosures
Masarova:MorphoSys US: Membership on an entity's Board of Directors or advisory committees. Kadia:Amgen, Inc.: Research Funding; Sanofi-Aventis: Consultancy; Daiichi Sankyo, Genentech, Inc., Genzyme, Jazz Pharmaceuticals, Liberum, Novartis, Pfizer, PinotBio, Inc, Pulmotect, Inc, Sanofi-Aventis, Servier: Consultancy; Astellas Pharma Global Development: Research Funding; GenFleet Therapeutics: Research Funding; Iterion: Research Funding; Cyclacel: Research Funding; Hikma Pharmaceuticals: Speakers Bureau; Agios: Consultancy; Genentech: Consultancy, Research Funding; AstraZeneca: Research Funding; Ascentage Pharma Group: Research Funding; Biologix, Cure, Hikma Pharmaceuticals: Speakers Bureau; Glycomimetics: Research Funding; Delta-Fly Pharma, Inc.: Research Funding; Janssen Research and Development: Research Funding; Liberum: Consultancy; AbbVie, Amgen, Inc, Ascentage Pharma Group, Astellas Pharma Global Development, Astex, AstraZeneca, BMS, Celgene, Cellenkos Inc, Cyclacel, Delta-Fly Pharma, Inc, Genentech, Inc., Genfleet, Glycomimetics, Iterion, Janssen Research and Development: Research Funding; Genzyme: Honoraria; Jazz Pharmaceuticals, Pfizer, Pulmotect, Inc, Regeneron Pharmaceuticals, SELLAS Life Sciences Group: Research Funding; Cure: Speakers Bureau; Celgene: Research Funding; Cellenkos Inc.: Research Funding; Regeneron Pharmaceuticals: Research Funding; Pulmotect, Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Servier: Consultancy; Pinotb-Bio: Consultancy; BMS: Consultancy, Research Funding; Astex: Honoraria; SELLAS Life Sciences Group: Research Funding. DiNardo:ImmuniOnc: Honoraria; BMS: Honoraria; AbbVie/Genentech: Honoraria; Novartis: Honoraria; Servier: Honoraria; Astellas: Honoraria; Fogham: Honoraria; Notable Labs: Honoraria; Takeda: Honoraria; Schrödinger: Consultancy. Bose:GSK, Novartis, Karyopharm, AbbVie, Pharma Essentia, Jubilant, Morphic: Honoraria; Kartos, Telios, Ionis, Disc, Janssen, Geron: Research Funding; Incyte, BMS, CTI, Morphosys, Blueprint, Cogent, Sumitomo: Honoraria, Research Funding. Pemmaraju:Neopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Magdalen Medical Publishing: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Aplastic Anemia & MDS International Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Curio Science: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Menarini Group: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Physician Education Resource (PER): Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PeerView Institute for Medical Education: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Patient Power: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Consultancy, Membership on an entity's Board of Directors or advisory committees; CareDx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; ASCO Cancer.Net Editorial Board: Other: Leadership; ImmunoGen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ClearView Healthcare Partners: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Protagonist Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Harborside Press: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medscape: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; United States Department of Defense (DOD): Research Funding; ASH Committee on Communications: Other: Leadership; Karger Publishers: Other: Licenses; Novartis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cimeio Therapeutics AG: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; OncLive: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pacylex: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CancerNet: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Intellisphere: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dava Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Imedex: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Aptitude Health: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; National Institute of Health/National Cancer Institute (NIH/NCI): Research Funding; HemOnc Times/Oncology Times: Other: Uncompensated; Bristol Myers Squibb Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Loghavi:Caris Diagnostics: Consultancy; Blueprint Medicine: Consultancy; Abbvie: Consultancy; Gerson Lehrman Group: Consultancy; QualWorld: Consultancy; Guidepoint: Consultancy; Recordati/ EUSA Pharma: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Research Funding; Amgen: Research Funding; Abbvie: Current equity holder in publicly-traded company. Törngren:Active Biotech: Current Employment. Bhalla:Foghorn Therapeutics Inc.: Research Funding.
