Evaluation of the Lethal Activity and Its Mechanism of Tasquinimod in Advanced Myeloproliferative Neoplasm (MPN) in Blastic Phase

S100A8 (A8) and S100A9 (A9) are low molecular weight, pro-inflammatory proteins that belong to the S100 protein family known as calprotectins. In response to environmental stimuli or cell damage, A8 and A9 are released into the extracellular space and plasma, where they interact with TLR4 (Toll like receptor 4), RAGE (receptor for advanced glycation end products) and CD33. Following homo- or hetero-dimerization, A8 and A9 act as Ca++ biosensors and activate NADPH oxidase, stimulating ROS production, which activates the NLRP3 inflammasome. Through TLR4, A8 and A9 activate NFkB and MAPK pathways and amplify the inflammatory response and induce inflammatory cytokines, including TNFα and IL-6. A8 and A9 have been shown to be highly expressed in AML and are associated with poor prognosis. In the murine JAK2-V617F-driven primary myelofibrosis (PMF) model, tasquinimod (TQ, Active Biotech), an orally active, quinoline-3-carboxamide immune-modulatory agent was shown to bind A9, inhibiting its interaction with TLR4 and RAGE receptors. Notably, TQ was shown to reduce myeloproliferation, splenomegaly and bone marrow fibrosis in JAK2-V617F mice. In the present studies, we determined in vitro and in vivo efficacy of TQ against cellular models of advanced MPN and patient-derived (PD) CD34+ blastic phase (BP, > 5% blasts in PB) MPN cells. Treatment with TQ (10 to 30 µM for 96 hours) induced significant loss of viability (20-30%; p < 0.05) in the MPN-AML SET2, HEL92.1.7 cell lines and PD MPN-BP cells, but not in normal CD34+ progenitor cells. In HS5 stromal cells, following treatment with TQ (20 µM), RNA-Seq analysis showed positive enrichment of the mRNA gene-set of TNFA Signaling via NFKB, with log2 fold-induction of IL6, LIF, IL1A, and CXCL2 but depletion of CCL2 and CXCL12 expressions. In PD MPN-AML cells, exposure to TQ (20 µM for 16 hours) caused negative enrichment of the gene-sets of MYC and E2F targets, inflammatory response, TORC1 and IL6-JAK-STAT3 signaling. This involved log2 fold-decline in MYC, BCL2, CDK6, MPL, CCND1 and myeloperoxidase (MPO), but induction of GFI1 and p21 mRNA. Mass spectrometry of PD, post-MPN sAML cells following TQ treatment demonstrated depletion of proteins involved in signal transduction, cytokine signaling and transcription. TQ treatment of PD, post-MPN sAML cells, also significantly altered cytokine protein expression levels, including depletion of IL-6, IL8/CXCL8, and CCL2/MCP1 but induction of TNFα. Following TQ treatment (20 µM for 48 hours), CyTOF analysis on PD, post MPN sAML cells showed that in phenotypically defined CD34+ stem/progenitor cells, with high levels of CLEC12A, CD99 and CD123 but low expression of CD11b, the protein expressions of A8, A9, p-ERK, MPO, CXCR4, Cyclin D1, PU.1 and Ki67 were reduced, whereas the levels of GFI1, p21 and cleaved PARP were increased. Importantly, cotreatment with TQ (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, represented by delta synergy scores of >1.0 (by the ZIP method). We next conducted a domain-specific epigenetic target-focused CRISPR screen in SET2 cells to agnostically determine druggable co-dependencies with TQ treatment. This identified the arginine methyltransferase CARM1 and the histone demethylase JMJD6 as TQ-co-dependencies, as well as HDAC3 and NSD2 as TQ co-enrichments. Co-treatment with TQ and RGFP966 (HDAC3i) induced synergistic lethality in post-MPN sAML cells. Treatment of mouse progenitors expressing JAK2-V617F and TP53-knockout transplanted in lethally irradiated, immune replete JAXBOY mice with TQ and/or OTX015 significantly reduced leukemia burden in the mice. Furthermore, monotherapy with TQ (10 or 30 mg/kg/day, by oral gavage), of NSG mice engrafted with MPN-AML cells (mutant (mt) CALR and mtTERT) significantly improved survival without inducing host toxicity. Compared to single agent treatment, cotreatment with TQ and ruxolitinib (30 mg/kg/day) or OTX015 (30 mg/kg/day) also significantly improved survival of the NSG mice. These findings demonstrate the pre-clinical efficacy of TQ and/or JAKi or BETi or with novel agents identified here in advanced MPN and MPN-AML cells. They also create the rationale to further interrogate the pre-clinical efficacy of the TQ-based combinations against cellular models of advanced MPN.