UNC2025, a Small Molecule MerTK and Flt3 Tyrosine Kinase Inhibitor, Decreases Disease Burden, Prolongs Survival, and Promotes Sensitivity to Chemotherapy in Xenograft Models of Acute Leukemia
Authors: DeRyckere Deborah , Lee-Sherick Alisa B. , Hill Amanda A. , Page Lauren , Wang Xiaodong , Frye Stephen V. , Earp H. Shelton , Graham Douglas K. 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) and relapsed or refractory acute lymphoblastic leukemia (ALL) are extremely difficult to treat and new therapies are needed to improve survival and decrease toxicities associated with conventional therapy. The MerTK receptor tyrosine kinase is aberrantly expressed in 30% of pediatric B-ALL patient samples, 50% of pediatric T-ALL samples, and 80-90% of pediatric and adult AML samples and inhibition of MerTK using shRNA prolonged median survival time in ALL and AML xenograft models. These data validate MerTK as a potential therapeutic target in acute leukemias. In AML, internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (Flt3) receptor are observed in ~20-30% of adult and ~15% of pediatric AML patient samples and are associated with poor prognosis. Flt3 inhibitors have demonstrated therapeutic efficacy in clinical studies but resistance to current Flt3-targeted TKIs and unacceptable adverse side effects have been reported, indicating a need for new therapeutic agents directed against Flt3. Here we report a novel dual MerTK/Flt3-selective small molecule TKI as a potential therapy for acute leukemias with MerTK expression and/or activating Flt3 mutation. UNC2025 mediated potent inhibition of MerTK and Flt3 in enzymatic (Ki = 0.16 nM and 0.59 nM, respectively) and cell-based (IC50 = 2.7 nM and 14 nM, respectively) assays and had limited off-target activity against 300 other kinases. Treatment with 100-200 nM UNC2025 inhibited MerTK-dependent pro-survival signaling pathways, induced apoptosis in 40-90% of cells, and reduced colony-forming potential by 80-100% in leukemia cell line cultures. Similarly, a MerTK-expressing leukemic patient sample exhibited reduced MerTK-dependent pro-survival signaling, induction of apoptosis, and decreased colony formation in semi-solid medium in response to UNC2025. In contrast, a MerTK-negative leukemic patient sample did not respond to treatment, suggesting that UNC2025-mediated anti-leukemia effects are a consequence of MerTK inhibition. UNC2025 is orally bioavailable and inhibits MerTK phosphorylation/activation in leukemic blasts in the bone marrow. Treatment with UNC2025 resulted in a dose-dependent reduction in tumor burden and increased median survival from 27 days after inoculation with tumor cells to 70 days in an orthotopic B-ALL xenograft model of minimal residual disease (p < 0.0001) and from 27.5 to 45 days in a similar model of existent disease (p < 0.0001). In a MerTK-expressing patient-derived xenograft (PDX) model of AML, short-term treatment with UNC2025 resulted in a significant decrease in peripheral disease burden (9.3% blasts versus 35.6% for vehicle-treated animals, p = 0.0225) and both the fraction of splenic blasts (28.5% versus 75.7%, p = 0.0005) and spleen mass (170 mg versus 834 mg, p < 0.0001) were dramatically decreased. UNC2025 was even more effective in a FLT3-ITD mutant, MerTK-expressing AML PDX model. In this case, treatment with UNC2025 resulted in stable disease regression (0.24% peripheral blasts after 4 weeks of treatment versus 11.4% pre-treatment, p = 0.0094) and prolonged median survival from 16 days post-treatment to > 107 days (p = 0.0114). In addition, in the B-ALL xenograft model, treatment with UNC2025 and methotrexate, a chemotherapy currently in clinical use for treatment of pediatric ALL, resulted in reduced tumor burden and increased tumor-free survival relative to mice treated with either agent alone, suggesting that addition of UNC2025 to standard therapies may be a particularly effective treatment strategy and/or may allow for chemotherapy dose-reduction and decreased toxicity. The very high potency, relative selectivity, oral bioavailability, and target inhibition and therapeutic efficacy mediated by UNC2025 in murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of patients with acute leukemia.

Disclosures
DeRyckere: Meryx, Inc.: Equity Ownership. Wang:University of North Carolina Chapel Hill: UNC2025 Patents & Royalties; Meryx, Inc.: Equity Ownership. Frye:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Earp:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Graham:Merys, Inc.: Equity Ownership.