Combining mertk inhibitor mrx-2843 with venetoclax and 5-azacitidine in AML Free

Introduction: Acute myeloid leukemia (AML) is the most aggressive form of leukemia, and therapies that can sustain long-term cures in AML patients remain elusive. Venetoclax, a BCL2 inhibitor, and azacitidine, a DNA methyltransferase inhibitor, are commonly administered together in newly diagnosed AML patients who are not candidates for intensive chemotherapy, including elderly patients. While many patients have initial responses, long-term sustained remission is rare. Hence, there is an urgent need for new therapies that can significantly improve clinical outcomes in AML. We identified MERTK (MER Receptor Tyrosine Kinase) as a novel therapeutic target in AML. MERTK is aberrantly expressed in over 80% of AML patient samples and MERTK inhibition is therapeutically effective against AML cells in vitro and in vivo. Here we describe development of a novel therapy for AML that combines MRX-2843 (an orally bioavailable dual MERTK and FLT3 inhibitor) with venetoclax and azacitidine to provide enhanced therapeutic effects.

Methods:

MERTK expressing human AML cell lines (NOMO-1, OCI-AML5, KG-1) were treated in vitro with MRX-2843, venetoclax, and/or azacitidine single agents and combinations (venetoclax/azacitidine or MRX-2843/venetoclax/azacitidine) and relative cell numbers were determined using CellTiter-Glo assay. Drug interactions were assessed by mathematical modeling using the fractional product method. Alternatively, cells were stained with Annexin-V and propidium iodide, and apoptotic and dead cells were detected by flow cytometry. For in vivo studies, xenografts were established by intravenous injection of KG-1 cells into NOD-SCID-gamma mice. Mice were randomized to groups (n=10-12 per group) and drug treatments were initiated 24-27 days after leukemia inoculation. MRX-2843 (65 mg/kg) and venetoclax (40 mg/kg) were administered once daily by oral gavage, and azacitidine (2.5 mg/kg) was administered once daily during the first 5 days of each 28-day treatment cycle by intraperitoneal injection. Mice were treated for a total of 150 days encompassing 5 consecutive 28-day cycles. Health status was monitored and survival was determined. Two independent studies were performed. In the second study, bone marrow disease burden (% human CD45+ cells) was assessed on treatment days 28 and 73 using flow cytometry (n=3-6 per group).

Results:

In cultures of all 3 AML cell lines, treatment with the 3-drug combination (MRX-2843/venetoclax/azacitidine) reduced cell numbers compared to treatment with MRX-2843 monotherapy or standard-of-care venetoclax/azacitidine alone. For instance, in KG-1 cultures, treatment with MRX-2843 or venetoclax/azacitidine reduced cell densities by 55±5% and 60±3%, respectively, compared to vehicle while the triple combination mediated a 96±1% reduction (p<0.0001). Mathematical modeling revealed that the interaction between MRX-2843 and venetoclax/azacitidine was synergistic in 2 of the 3 cell lines and additive in the other. Treatment with the 3-drug combination also enhanced leukemia cell killing compared to MRX-2843 or venetoclax/azacitidine in all 3 cell lines. For example, in OCI-AML5 cultures, treatment with the 3-drug combination induced apoptosis and cell death in 80±1% of cells, compared to 34±2% (p=0.0036) and 71±0.5% (p=0.0212) in MRX-2843-treated and venetoclax/azacitidine-treated cultures, respectively.

These findings translated to a mouse AML xenograft model. In 2 independent studies, the triple combination significantly prolonged mouse survival (59.1% survival after 150 days of treatment) compared to MRX-2843 monotherapy (0% survival, 76.5 days median survival, p < 0.0001) or venetoclax/azacitidine (4.6% survival, 104.5 days median survival, p < 0.001). Furthermore, the fraction of leukemic blasts in the bone marrow was significantly reduced after the first treatment cycle in mice treated with the 3-drug combination (8.5±5%, n=4) compared to mice treated with vehicle (67±8%, n=5, p<0.001), MRX-2843 (41±9%, n=6, p = 0.0451) or venetoclax/azacitidine (40%±8%, n=6, p=0.0482).

Conclusions:

Treatment with MRX-2843 sensitized AML cells to standard-of-care venetoclax/azacitidine, leading to increased AML cell death, more effective targeting of AML cells in the bone marrow, and prolonged survival in mouse models. These findings implicate combined treatment with MRX-2843, venetoclax and azacitidine as an effective therapeutic strategy with potential to improve outcomes for patients with AML.