Novel MET Inhibitors Provide a Therapeutic Opportunity for AML By Overcoming the Protective Action of the Bone Marrow Niche
Authors: Bueno Maura Lima Pereira , Ayemoba Charles , Di Staulo Anna M , Zhang Sen , Niemann Fernanda Soares , Amôr Nádia Ghinelli , Santos Irene , Duarte Adriana da Silva Santos , Bastos Audrey , Saad Sara Teresinha Olalla , Pinho Sandra , Roversi Fernanda Marconi 
Published: December 2024

Acute Myeloid Leukemia (AML) is an aggressive neoplasm triggered by the uncontrolled growth of abnormal myeloid cells. This translates to poor patient outcomes with significant challenges in achieving a cure due to resistance to standard chemotherapy and frequent relapse. Targeting signaling pathways that support AML-initiating cells, known as leukemic stem cells (LSCs), such as the HGF/MET-hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (MET)-pathway, holds promise for LSC elimination and improving AML therapy. HGF is secreted by mesenchymal stromal cells (MSCs) in the bone marrow (BM) tumor microenvironment (also known as niche), and interacts with the MET receptor, activating pro-survival pathways in AML cells. Yet, developing novel drugs that specifically target the HGF/MET interaction is a challenge, and overcoming the protective influence of the BM niche remains a persistent hurdle. Given the pivotal role of HGF/MET in AML prognosis and progression, we investigated the effects of three promising novel MET inhibitors, B2000, B6000, and C1000 (Brigham University, patent WO 2011/127192 A2).

To evaluate the therapeutic potential of the MET inhibitors, we first assessed the cytotoxicity of these compounds. The inhibitors promoted apoptosis (Annexin V+) in primary bone marrow mononuclear cells (BMNCs) from AML patients (p=.0059) without significant effect on healthy cells. In AML cell lines, Molm-13 (FAB M5a, MLL-AF9-mutated) and OCI-AML3 (FAB M4, NPM1-mutated), treatment with MET inhibitors resulted in low IC50 values (&lt;10µM) and increased apoptosis rates (50% increase, p&lt;.0001), indicative of a potent activity. Moreover, an aggressive MLL-AF9-driven AML model was employed to evaluate the efficacy of the MET inhibitors. The treatment reduced in 50% and 92% the proliferation of total AML cells and the phenotypic LSCs (Lineage−CD127−Sca1MLL-AF9-GFP+c-KithighCD34+CD16/32high) in vitro (p&lt;.0001), respectively. To validate these findings in a pathophysiological setting, leukemic C57BL/6 mice bearing MLL-AF9-driven AML received daily intraperitoneal injections of the MET inhibitors (10mg/kg) for 5 days. Strikingly, the C1000 compound demonstrated the most potent effect, significantly inhibiting in 70% the proliferation of AML cells and LSCs in the BM, spleen, and peripheral blood (p&lt;.0001).

Building on these findings, we investigated the effects of these MET inhibitors within the BM niche, where the microenvironment benefits AML cell survival. Molm-13 and OCI-AML3 cells were cocultured with primary BM MSCs to simulate the BM niche conditions. Remarkably, after treatment with MET inhibitors, the HGF signal was reduced on the AML cell surface, indicating disruption of the HGF/MET interaction. To gain a more nuanced understanding of these effects, we employed a 3D vascular and perivascular niche-mimicking co-culture model with AML cells. The treatment successfully overcame the protective effect of the niche cells (endothelial cells and MSCs), as evidenced by a 71% reduction in Molm-13 and blasts from AML patients (p&lt;.0001). Finally, to explore the practical application of the C1000 compound for a combinatory therapy approach, AML cell lines were treated with C1000 alone or in combination with Venetoclax, a BCL2 inhibitor currently used for the treatment of AML patients who are ineligible for intensive chemotherapy. Synergy analysis performed using the Synergy Finder package in R revealed a synergistic interaction between C1000 and Venetoclax compounds (Bliss, ZIP, Loewe, HAS synergy scores &gt;10). Taken altogether, our data demonstrate that MET inhibitors exhibit potent anti-neoplastic activity against aggressive AML models without harming healthy cells. Additionally, C1000 synergizes with Venetoclax compound, suggesting a promising therapeutic opportunity for combined chemotherapy regimens.

Funding: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0, 2022/14101-2; CNPq 303405/2018-0.